Maxillofac Plast Reconstr Surg 2014;36(3):103-110 http://dx.doi.org/10.14402/jkamprs.2014.36.3.103 ISSN 2288-8101(Print) ISSN 2288-8586(Online)

Original Article

Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy Studies on Processed Tooth Graft Material by Vacuum-ultrasonic Acceleration Eun-Young Lee, Eun-Suk Kim1, Kyung-Won Kim Department of Oral and Maxillofacial Surgery, Medical Research Institute, College of Medicine, Chungbuk National University, 1 Department of Oral and Maxillofacial Surgery, Jukjeon Dental Hospital, College of Dentistry, Dankook University

Abstract Purpose: The current gold standard for clinical jawbone formation involves autogenous bone as a graft material. In addition, demineralized dentin can be an effective graft material. Although demineralized dentin readily induces heterotopic bone formation, conventional decalcification takes three to five days, so, immediate bone grafting after extraction is impossible. This study evaluated the effect of vacuum ultrasonic power on the demineralization and processing of autogenous tooth material and documented the clinical results of rapidly processed autogenous demineralized dentin (ADD) in an alveolar defects patient. Methods: The method involves the demineralization of extracted teeth with detached soft tissues and pulp in 0.6 N HCl for 90 minutes using a heat controlled vacuum-ultrasonic accelerator. The characteristics of processed teeth were evaluated by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). Bone grafting using ADD was performed for narrow ridges augmentation in the mandibular area. Results: The new processing method was completed within two hours regardless of form (powder or block). EDS and SEM uniformly demineralized autotooth biomaterial. After six months, bone remodeling was observed in augmented sites and histological examination showed that ADD particles were well united with new bone. No unusual complications were encountered. Conclusion: This study demonstrates the possibility of preparing autogenous tooth graft materials within two hours, allowing immediate one-day grafting after extraction. Key words: Autogenous, Demineralized dentin, Vacuum, Ultrasonic, Energy dispersive X-ray spectroscopy

and hydroxyapatite (HA) crystals, are also used for bone

Introduction

regeneration. Dentin is a potentially valuable graft material.

Autogenous bone grafts are extensively used to augment

Initially, particulate dentin (tooth ash) was used as a graft

severely resorbed alveolar ridges, although other materials,

material, because hydroxyapatite is the main ingredient

such as, undecalcified, allogeneic devitalized bone, frozen

of teeth. Because it is bioresorbable, particulate tooth ash

bone, processed xenogeneic bone, deproteinized bone,

can also be used as an alternative graft material for im-

RECEIVED February 9, 2014, REVISED February 28, 2014, ACCEPTED April 21, 2014 Correspondence to Eun-Young Lee Department of Oral and Maxillofacial Surgery, Medical Research Institute, College of Medicine, Chungbuk National University 52 Naesudong-ro, Heungdeok-gu, Cheongju 361-763, Korea Tel: 82-43-269-6294, Fax: 82-43-269-6779, E-mail: [email protected] Copyright © 2014 by The Korean Association of Maxillofacial Plastic and Reconstructive Surgeons. All rights reserved. CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

plants[1]. Demineralized dentin has recently been used for

bone regeneration using the novel tooth grafts.

the reconstruction of alveolar bone[2]. In fact, the chemical composition of dentin is similar to that of bone. It is composed of a hydrated organic matrix consisting mainly of type I collagen and an inorganic reinforcing phase of carbonated apatite. Dentin is composed of inorganic (70%∼

Materials and Methods 1. Materials

75%), organics (20%), and water (10%), whereas in alveolar

Chungbuk National Hospital BioBank provided tooth

these percentages are 65%, 25%, and 10%, respectively[3].

samples (n=8) for this research, and all donors provided

On the other hand, decalcified dentin is a composite matrix

informed consent and the study was approved by IRB.

of type I collagen and growth factors. Furthermore, dentin

The samples were randomly allocated to an autogenous

and bone demineralization increases osteoinductivity, and

demineralized dentin (ADD) group (n=5) and an untreated

decreases antigenicity. The bone-inducing property of den-

group (n=3). Human extracted teeth were washed in a

tin was discovered in 1967 when demineralized dentin ma-

solution of 0.9% NaCl after soft tissue and pulp removal

trices (DDM) of rabbit were found to induce bone for-

and stored at 󰠏70 C.

mation in intramuscular tissues[4].

o

In the ADD group, remnant tissues around the roots

A problem with demineralized dentin as a graft material

of extracted teeth were removed using a surgical blade

is the time it takes to decalcify and produce as a bone

or periosteal elevator. A dental handpiece was used to

graft material from teeth. In particular, tooth blocks require

remove pulp tissue from the pulp chamber and root canals

more processing time than tooth chips or powder. The con-

and perfuse the reagent effectively. In addition 0.3 mm

ventional method requires 10 days or more to complete

diameter holes were made along the entire tooth surface

because bone transplant material must be prepared using

using a 330 bur (Fig. 1). The tooth was placed in a sterile

extracted teeth. This requires transport to a processing com-

solution for 10 minutes in the vacuum ultrasonic system.

pany, and at least 10 hours is required to demineralize a

Teeth were demineralized in a 0.6 N solution of HCl for

tooth block. As a faster alternative method, we developed

90 minutes in the vacuum ultrasonic machine (VacuaSonic ),

TM

TM

a vacuum-ultrasonic accelerator (VacuaSonic ; Cosmobio-

and washed for twice for 15 minutes in deionized sterile

medicare, Seoul, Korea) based method that can be performed

water or phosphate buffered saline (PBS) solution. The

in clinics. The whole process can be completed within two

entire processing time was less than 2 hours (Fig. 2). After

hours regardless of the form (powder, chip or block) used.

removing the PBS solution, processed teeth graft materials

The new method utilizes ultrasonication and a vacuum.

were packaged into a sterile tube.

Ultrasonication uses for cleaning, but because of the hard-

In the untreated group, remnant tissues around the roots

ness of teeth, it is difficult to clean hidden surfaces (dentinal

were removed using a surgical blade or periosteal elevator,

tubules). In addition, it produces trapped air bubbles in dentinal tubules. We used a periodic vacuum system and specific ultrasonic frequency to solve these problems. The chemical treatment employed with ultrasonication included a vacuum, which noticeably improved efficiency, and markedly reduced overall treatment time[5]. We studied the ultra-structure and chemical components of newly processed tooth graft material by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). A clinical case revealed the ability of the graft materials produced to regenerated bone after implant surgery. Here we report preliminary results obtained from our experimental studies and a clinical case of alveolar

Fig. 1. Processed teeth (autogenous demineralized dentin).

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

105

Instruments, Tonawanda, NY, USA), after coating samples

then the teeth were washed in a saline solution. Two groups of tooth samples were prepared in the form of 2 mm cubes for SEM and EDS studies. Cubes were removed from the tooth necks with a #15 blade (ADD) and a high speed bur (untreated teeth).

with a gold-palladium alloy.

4. Energy dispersive X-ray spectroscopy To determine whether a change in mineral components occurred during processing, EDS was performed using the

2. Measurements of wet tooth weight

TEAM

To quantify weight reduction, weights were measured

TM

system (Bruker Co., Ewing, NJ, USA). To de-

termine internal mineral concentrations, processed tooth materials and unprocessed teeth were checked for calcium

before and after processing in ADD group.

concentration (wt%) at a depth of 300 to 600 micrometers

3. Scanning electron microscopy

from tooth surface.

Observations of ADD material and untreated tooth surfaces were performed optically using a scanning electron

5. Clinical case

microscope, in secondary electron mode. SEM studies were

A 45-year-old male patient visited Chungbuk National

performed using a Nanoscope IIIa instrument (Digital

University Hospital for extraction of #48 tooth. Radiological and clinical examinations revealed an impacted third molar, a periapical and a periodontal lesion of #47, and an edentulous alveolar ridge on #44 with narrow ridge (Fig. 3).

Fig. 2. Autogenous demineralized dentin processing procedures (from extraction to graft).

Fig. 3. Panoramic view; pre-extraction of #47, 48.

Fig. 4. Pre-operative computerized tomography. (A) #44 extracted area (arrow: narrow ridge). (B) #47 extracted area (arrow: narrow ridge).

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

Therefore, surgical extraction of #47 and 48 and implant

USA) (Fig. 6). After suturing without tension, antibiotics

placement on #44 and 47 with ADD were planned. #47

and anti-inflammatory analgesic were administered and the

and 48 teeth were extracted before surgery, and block-type

patient gargled with saline.

autogenous tooth graft materials were produced in a clinic. For personal reasons, surgery was delayed for six months, o

and processed materials were stored at 󰠏70 C in a freezer. Preoperative computer tomography was performed (Fig. 4). The patient was prepared and sterilely draped for intraoral surgery. Significant narrow ridges on #44 (2 mm)

Results 1. Wet tooth weights The weights of teeth before processing were measured

and #47 (4 mm) edentulous sites were noted. Implants

at an average 0.38 g (range, 0.23∼0.53 g). The weights

were placed on the #44 (diameter 4.0 mm, length of 10

of teeth after processing averaged 0.20 g (range, 0.13∼0.32

mm; TSIII; Osstem, Seoul, Korea) and #47 area diameter

g), 43.4% average reduction (Table 1).

4.5 mm, length of 10 mm; TSIII). Exposed implant threads were noted due to the narrow ridges (Fig. 5). An autoge-

2. Scanning electron microscopy

nous tooth chip was prepared with the bone mill and

Fig. 7 shows SEM images of the processed dentin. The

placed on the buccal side of alveolar ridge. To increase

external aspects of ADD surfaces with open dentinal tu-

volume, a block type of ADD was transplanted upwards

bules were visualized. Tubule sizes increased after

autogenous tooth chip with resorbable collagen membrane

processing. SEM visualized processed dentin surfaces with

(CyADDlast; Osteogenic Biomedical Inc., Lubbock, TX,

widened dentinal tubules.

Fig. 5. Intra-operative photos. Showing are narrow ridges and exposure implant threads. (A, B) #44 extracted area (circle: narrow ridge, arrow: exposed fixture). (C, D) #47 extracted area (circle: narrow ridge, arrow: exposed fixture).

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

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Fig. 6. Autogenous demineralized dentin chip and block graft. (A) #44 extracted area (arrow: particulated processed tooth graft). (B) #44 extracted area (arrow: block processed tooth graft). (C) #47 extracted area (arrow: particulated processed tooth graft). (D) #47 extracted area (arrow: block processed tooth graft). Table 1. Autogenous demineralized dentin weight measurements Patient Donor age (yr)/sex No. 1 2 3 4 5

52/male 40/male 18/female 22/male 56/male

Pre-processing (g)

Post-processing (g/%)

0.53 0.28 0.51 0.23 0.35

0.32/60 0.16/57 0.26/51 0.13/57 0.20/58

Fig. 8. Scanning electron micrograph of an untreated dentin surface (bar scale 20 μm).

We paid attention to unprocessed dentin on the surfaces. Dentinal tubules were not seen. Fig. 8 shows a dentin surface with a smear layer and ground tooth particles proFig. 7. Scanning electron micrograph of a dentin surface after the Autogenous demineralized dentin processing (bar scale 20 μm).

duced by a high speed bur. In untreated teeth smear layers were composed of small particles of dentin and dendrites

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

were higher than those of ADD. Mineral concentrations,

covered dentin surface.

3. Elemental analysis by energy dispersive X-ray spectroscopy The calcium and phosphate levels of untreated teeth

were significantly lower for ADD. Changes in calcium level in ADD grafts were observed. The calcium concentration of ADD was 6.52 wt% whereas that of normal dentin was 31.17 wt% at a depth of 300∼600 micrometers from external surfaces (Table 2), indicating that 79% of the calcium had been removed (Fig. 9, 10).

Table 2. Calcium concentration of processed (autogenous demineralized dentin) and unprocessed teeth Variable Processed tooth (n) 1 2 3 4 5 Unprocessed tooth (n) 1 2 3

Depth (μm)

Calcium level (wt/%)

307.6923 448.7179 641.0256 384.6154 551.2821

7.14 4.45 5.24 7.42 8.34

411.9318 553.9773 696.0227

30.98 32.46 30.07

4. Clinical observations and histopathologic analysis In the described case, healing was uneventful. No significant complications, such as, infection, uncontrolled dehiscence, graft failure, or implant failure occurred. Six months after surgery, good bone healing and bone remod-

Fig. 9. Energy dispersive X-ray spectroscopy of a dentin surface after the autogenous demineralized dentin processing.

eling were observed. Further examination of computer tomographic views was performed to confirm bone healing at ADD treated defect sites (Fig. 11).

Fig. 10. Energy dispersive X-ray spectroscopy of an untreated dentin surface.

Fig. 11. Post-operative computerized tomography. Six months after the autogenous demineralized dentin graft, good bone formation is seen. (A) #44 extracted area. (B) #47 extracted area.

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109

dentin proteins, such as, osteocalcin, osteonectin, phosphoprotein, and sialoprotein are involved in bone calcification[8,9]. Furthermore, both mature and immature types of BMPs-2 are found in human dentin[10]. Decalcified dentin supports heteroinduction in experimental and clinical studies[4,11-14]. Decalcified dentine is penetrated and resorbed more slowly than decalcified bone, because it is denser and has neither old vascular channels nor marrow spaces[4]. For the same reason, the decalcification time of teeth is greater than that of bone. Long processing times for teeth are problematic clinically. Moreover, excessive acid exposure might diminFig. 12. Histological finding of autogenous demineralized dentin at 6 months after implantation (H&E, ×200; open arrows: new bone formation, closed arrows: processed tooth graft material). New bone formation around processed tooth material and resorption of grafted materials.

ish BMP retention due to diffusional losses and graft potency. As described by Pietrzak et al.[15], the demineralization of bovine cortical bone using 0.5 N HCl occurred within 90 minutes and peak BMP-7 levels were reached at that point, and then fell. To overcome this prob-

For histopathologic examination, a bone specimen was

lem, we developed a rapid processing method using a

sampled at ADD treated site. The specimen showed new

vacuum-ultrasonic system. Our SEM and EDS studies con-

bone formation and bone remodeling. In addition, apposi-

firmed the effective demineralization of teeth within two

tional bone growth and ADD particles resorption were ob-

hours. SEM analysis of newly decalcified dentin (ADD)

served (Fig. 12).

revealed dentinal tubules, which we believe act as a network for the diffusion of nutrients after grafting. Type I collagen fiber accounts for 90% of the organic matrix, and

Discussion

is important in calcification. Demineralization of dentin

The present study documented the characteristics and

and bone increases osteoinductivity and decreases anti-

the ability of alveolar bone reconstruction using rapidly

genicity[16]. Ultrasonic treatment creates cavitation, a high

demineralized dentin (ADD) as a bone graft material.

energy phenomenon, inside an object by repeated com-

Dentin is an acellular collagen-rich tissue matrix without

pression and expansion[17]. When this cavitation is present

vessels, whereas bone is a cellular tissue with vessels.

on the surface of an object, the energy released activates

However, dentin and bone have similar compositions, that

a reaction between the reagent and the object. In order

is, 10% body fluid, 20% organic materials, and 70% minerals

to use the ultrasonic system, we considered two factors;

(mainly HA), and contain bone morphogenetic proteins

temperature and air bubbles. Increased temperature during

(BMPs) that have the ability to induce bone and cartilage

processing affects the preservation of protein substance

in formation in non-skeletal sites[6,7]. The major compo-

in graft material. As air bubbles in dentinal tubules hinders

nent of the inorganic material of teeth contains several

rapid demineralization, periodic vacuum application was

types of calcium phosphate including HA. Earlier research

used to prevent bubble entrapment in dentinal tubules[5].

found that tooth ash can be used as graft material for the

The devised method facilitates clinical application be-

reconstruction of bone[1]. DDM performs better for bone

cause it can perform block processing within two hours.

induction than calcified dentin at four weeks after im-

If the operator chooses to use chip or powder, changes

plantation[4]. Demineralization of dentin increases the bio-

can be made during bone milling. In the described case,

availability of matrix-associated non-collagenous proteins,

we used block and chip (ADD) at no additional cost.

rendering these grafts osteoinductive. Non-collagenous

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Eun-Young Lee: Rapidly Processed Teeth Graft Materials

Conclusion

6.

The described vacuum-ultrasonic system enables tooth demineralization within two hours. This study shows that

7.

the developed processing method markedly reduces decalcification times. Furthermore, our high-resolution imaging

8.

and EDS studies confirmed the decalcification of dentin. In the clinical case, we observed good bone formation

9.

in narrow alveolar ridges. Finally, we conclude rapid preparation of autogenous teeth using the devised method makes one-day grafting possible.

10.

Acknowledgements

11.

This work was supported by a research grant of 12.

Chungbuk National University in 2012.

13.

References 1. Kim YK, Kim SG, Lee JG, Lee MH, Cho JO. An experimental study on the healing process after the implantation of various bone substitutes in the rats. J Korean Assoc Oral Maxillofac Surg 2001;27:15-24. 2. Chung JH, Lee JH. Study of bone healing pattern in extraction socket after application of demineralized dentin matrix material. J Korean Assoc Oral Maxillofac Surg 2011;37:365-74. 3. Min BM, editor. Oral biochemistry. Seoul: Daehan Narae Publishing; 2007. p.8-73. 4. Yeomans JD, Urist MR. Bone induction by decalcified dentine implanted into oral, osseous and muscle tissues. Arch Oral Biol 1967;12:999-1008. 5. Lee EY. Experimental study on processed teeth graft material with vacuum-ultrasonic accelerator [master’s thesis]. [Daejeon]:

14.

15.

16. 17.

Department of Medicine, Graduate School Chungnam National University; 2012. 48 p. Urist MR. Bone: formation by autoinduction. Science 1965; 150:893-9. Bilezikian JP, Raisa LG, Martin TJ. Principle of bone biology. Volume II. In: Heaney RP, editor. Calcium. Amsterdam: Elsevier; 2008. p.1697-710. Huggins C, Wiseman S, Reddi AH. Transformation of fibroblasts by allogeneic and xenogeneic transplants of demineralized tooth and bone. J Exp Med 1970;132:1250-8. Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S, Baylink DJ. Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin. J Bone Miner Res 1990;5: 717-23. Ito K, Arakawa T, Murata M, et al. Analysis of bone morphogenetic protein in human dental pulp tissues. Arch Bioceram Res 2008;8:166-9. Urist MR. Bone histogenesis and morphogenesis in implants of demineralized enamel and dentin. J Oral Surg 1971;29: 88-102. Nordenram A, Bang G. Reconstruction of the alveolar process by implantation of allogenic demineralized dentin. Oral Surg Oral Med Oral Pathol 1975;40:48-50. Movin S, Borring-Møller G. Regeneration of infrabony periodontal defects in humans after implantation of allogenic demineralized dentin. J Clin Periodontol 1982;9:141-7. Gomes MF, Abreu PP, Morosolli AR, Araújo MM, Goulart M. Densitometric analysis of the autogenous demineralized dentin matrix on the dental socket wound healing process in humans. Braz Oral Res 2006;20:324-30. Pietrzak WS, Ali SN, Chitturi D, Jacob M, Woodell-May JE. BMP depletion occurs during prolonged acid demineralization of bone: characterization and implications for graft preparation. Cell Tissue Bank 2011;12:81-8. Pashley DH. Dentin: a dynamic substrate--a review. Scanning Microsc 1989;3:161-74. Behrend O, Schubert H. Influence of hydrostatic pressure and gas content on continuous ultrasound emulsification. Ultrason Sonochem 2001;8:271-6.

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